Inhibition Of Endoplasmic Reticulum Stress Alleviates Secondary Injury After Severe Traumatic Brain Injury

BackgroundTraumatic brain injury(TBI)is a major socioeconomic problem and common cause of death and disability worldwide.What’s more,the mortality of severe TBI is up to 25%-50%.TBI can cause two types of neural tissue injury.The first is primary injury,which is mainly attributable to the initial physical forces applied to the brain at the moment of impact.The second type is secondary injury,there are many potential targets for intervention during this process.Apoptosis after traumatic brain injury has been shown to be a major factor influencing prognosis and outcome.Endoplasmic reticulum stress may be involved in mitochondrial mediated neuronal apoptosis.Therefore,endoplasmic reticulum stress has become an important mechanism of secondary injury after traumatic brain injury.Control of secondary brain injury is the key to severe TBI treatment,and to explore deep mechanisms around ERS to of secondary brain injury after severe TBI,making a great significance on prevention and treatment strategies.Method and PurposeAccording to lateral fluid percussion methord(LFP)made by Dixon and adopt the criteria for the classification of mechanical parameterst to estiblise severe TBI model in rats,to investigate the underlying mechanism linking apoptosis to TBI,we looked at the activation of the ER stress response,mitochondrial dysfunction as well as ROS production post-TBI.Furthermore,to investigate whether inhibition of ER stress could attenuate the severity of brain damage,mitochondrial dysfunction and ROS production post-TBI,a specific ER stress inhibitor,salubrinal,was administered before TBI.Results1.Elevated endoplasmic reticulum(ER)stress-related unfolded protein response(UPR)and activated the apoptosis ER stress response pathway post-TBI.To explore whether TBI induces ER stress and activates the UPR,we investigated the ER stress-related proteins,including phospho-PERK,PERK,phospho-eIF2a,eIF2a,ATF4,Spliced XBP-1,Cleaved ATF6 and GRP78.PERK and eIF2a phosphorylation was activated immediately at 1 h and peaked at 3 h post-TBI;ATF4 expression was induced at 3 h and peaked at 6 h post-TBI;Activation of Cleaved ATF6 was observed as early as 1 h and peaked immediately(3 h)post-TBI;Expression of Spliced XBP-1 was activated at 3 h post-TBI;GRP78 was induced and peaked at 3 h post-TBI.Taken together,these results suggest that intense TBI activated endoplasmic reticulum(ER)stress-related unfolded protein response(UPR),including three UPR transducer pathways:PERK-eIF2a-ATF4,XBP-1 and ATF6.Based on the above findings that TBI induced ER stress-related UPR signal transduction pathways,we next investigated whether TBI also activates the apoptosis ER stress response pathway.First,we detected expression of GADD153 and Caspase-12,the vital mediators of ER stress-induced apoptosis,GADD153 was activated at 3 h and peaked at 6 h,remained elevated at 12 h post-TBI;Caspase-12 was also significantly up-regulated at 3 h and peaked at 6 h,remained high to 24 h post-TBI;And the Caspase-12 activity has the similar consistent with the dynamic changes in the expression of Caspase-12 protein2.TBI induced mitochondrial injury and activated mitochondrial apoptosis pathwayTBI induced mitochondrial injury,led to decrease of mitochondrial membrane potential.We also investigated the release of Cytochrome C(Cyt C),the change of Bax/Bcl-2 ratio,Capsase-9 and-3 activity.Western blots showed a significant increase in the release of Cyt C into the cytosol,which increased beginning in 6 h and being for a period of 24 h post-TBI.Moreover,the abundance of Bax protein increased with a similar expression trend as pro-apoptotic Cyt C.Conversely,the expression of anti-apoptotic Bcl-2 decreased at the same time frame,suggesting an increase in the Bax/Bcl-2 ratio.Caspase-3 activity significantly increased at 6 h and peaked at 12 h post-TBI.These results implied that both ER stress response and mitochondrial apoptosis pathway were activated post-TBI;the ER stress response pathway was activated at the initial stage,while mitochondrial apoptosis pathway was activated a bit later.3.Sal alleviated brain damage,and mitigated both endoplasmic reticulum and mitochondrial damage post-TBIPretreatment with an ER tress inhibitor salubrinal(Sal)alleviated the pathological damage degree,oxidative stress,endoplasmic reticulum and mitochondria damage,as well as improved the nerve function score post severe TBI;Salubrinal via increasing the level of phosphorylation eIF2a to inhibit the expression of GADD153 and Caspase-12,then inhibited endoplasmic reticulum apoptosis pathway.Meanwhile,pretreatment with an ER tress inhibitor salubrinal(Sal)promoted recovery of mitochondrial protection function,inhibited the release of Cyt C into cytosol,decreased the Bax/Bcl-2 ratio,Capsase-9 and and-3 activation,finally reduced the occurrence of apoptosis post-TBI.ConclusionOur study provides evidence that TBI activated endoplasmic reticulum(ER)stress-related unfolded protein response(UPR),including three UPR transducer pathways:PERK-eIF2a-ATF4,XBP-1 and ATF6;both ER stress response and mitochondrial apoptosis pathway were activated post-TBI;the ER stress response pathway was activated at the initial stage,while mitochondrial apoptosis pathway was activated a bit later.More importantly,Sal pretreatment could not only alleviate the ROS production,the ultrastrutural damage of neurons ER and mitochondria,but also inhibit the ER stress response and mitochondrial apoptosis pathway at the different stage during secondary injury post-TBI.Thus,the amelioration of ER stress could be a novel strategy for preventing and treating during the secondary brain injury post-TBI.